BIOSENSORS IN PHARMACOLOGY
Актуальные публикации по вопросам современной медицины и здравоохранения.
by Ivan SENIN, Dr. Sc. (Chem.), Head of the Laboratory of Visual Reception, A. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
Monitoring of biological processes is essential for understanding the causes of diseases and finding methods for their therapy. One of the instruments for this are biosensors-sensitive elements recognizing biological objects. A prototype of such device has been created at A. Belozersky Institute of Physico-Chemical Biology. This device permits real-time studies of the most important class of receptor proteins regulating a majority of physiological processes in the organism. It is for this reason that these proteins serve as targets for almost half of the existing drugs used now. We hope that this approach will promote screening of numerous molecules, essential for detection of perspective drugs with preset characteristics.
TRANSMITTER PROTEINS
Perception of information from the environment is one of the key characteristics of living organisms, due to which they can adapt to the environment, find food, feel heat and cold. It is clear that the loss of capacity to correctly "read" external signals is fatal.
Humans, as other multicellular organisms, have two levels of signal perception. The organism as a whole gets information from the environment by means of vision, hearing, and other organs of senses. The cells which form the body, receive it only due to constant "intercourse" with each other. They use mainly the language of chemistry, represented by a variety of initial messengers ("communicators"), including hormones, neurotranmsmitters*, protein factors. Thus,
* Neurotransmitters (neuromediators) are biologically active chemicals, which help transmit an electric impulse from a nerve cell through synaptic space between neurons.--Ed.
Cell membrane section (scheme).
body cells do not live separately, their behavior depends on the presence of neighbors and is regulated by cell-to-cell contacts. They have to "deal" with each other so that their metabolism, proliferation (reproduction), and death, location in this or that organ or tissue, other functions and characteristics be governed primarily by the interests of the community. If it is healthy--its members are in harmony, cell proliferation and natural death (apoptosis) are balanced. If the regulation of these natural processes is impaired--predominance of apoptosis leads to tissue degeneration, while the victory of cell individualism and unlimited proliferation leads to the development of a malignant tumor, leading to death.
We face a reasonable question: how do cells "feel" the changes in the environment? External signals cannot penetrate into them directly, as they are protected by a plasmatic membrane: though it is thin, it serves as a sufficiently reliable barrier for many molecules, including the signal ones. However, in the majority of cases these molecules do not penetrate into the cell, but specifically react with its outer surface, to be more precise, with a special family of receptor proteins located on the external cellular membrane. The signal molecules just initiate the pulse transfer through it by stimulating the receptors in very low concentrations--10" M and lower.
There are several types of such receptors, but G-protein coupled receptors (GPCR) now attract the greatest attention; their number is over 800. All GPCR, despite their variety, are monomeric integral proteins, their polypeptide chain crosses seven times the cell membrane.
In the membrane GPCR react with molecules responsible for cell-to-cell communication, specifically, with hormones and neurotransmitters. When one of the receptors meets such a molecule, it transmits the signal through the membrane, which, in its turn, initiates an appropriate response of the cell.
This receptor type is particularly interesting for pharmacology due to its key role in cell-to-cell biochemical interactions responsible for regulation of functions important for health, including the heart and lung work, digestion and inflammatory reactions, as well as perception, memory, and even emotional sphere.
HOW ARE DRUGS CREATED?
Thus, we have presented a general picture of one of the main mechanisms due to which the host cells perceive external signals. It is clear that any disorder in the GPCR functioning leads to partial or complete loss of regulation of metabolic processes, which becomes a cause of grave diseases.
At the present stage of development of biology and medicine, new approaches are available for studies of normal processes of vital human activity and for active and controlled intervention in these processes in case of different pathologies. We should like to mention
G-protein-conjugated receptor.
Mechanism of G-protein-conjugated receptor stimulation.
here such trends of research as bioinformatics and bio-engineering, integrating in themselves the progress in various spheres of science, including mathematics, physics, chemistry, etc. However, despite the rapidly increasing scope of knowledge, the process of creation of sophisticated drugs, specifically those which use GPCR as targets, remains very expensive and labor-consuming: the creation of a drug takes 15 years, on average.
Why is the path to success so long? The matter is that at the first stage of search for potential drugs reacting with GPCR the effects on the target are studied on compounds chosen at random. The selective activities of hundreds of thousands and even millions of chemicals are tested. As a result, several of them are selected for subsequent evaluation of not only their effects on the target, but of their side effects too, such as toxicity, and other properties. And only after thorough testing and development of the technology of industrial synthesis, the concrete compound can be allowed for clinical trials.
Hence, it is obvious that the duration of drug creation largely depends on the stage when an enormous scope of chemicals is screened. This necessitates search for unusual approaches to monitoring and detection of biological processes at the stage of screening (primary selection) with minimal time outlays. One of the approaches to the development of appropriate technology is creation and use of the above-mentioned biosensors--sensitive elements recognizing different biological objects. In addition, the reactions are detected by a highly sensitive method--surface plasmon resonance* (SPR) spectroscopy. We shall speak about it below.
REAL-TIME MONITORING
A biosensor is an intricate technological device, in which the biological material, immobilized on solid phase surface, reacts to the presence of the test component and initiates the signal proportional to the concentration or amount of this component. The biosensor generally consists of two main "blocks": one for recognition and the other (instrumental) for transformation of the binding signal into some physical value. The SPR spectroscopy device is quite fit for the purpose: it realizes real-time monitoring of interactions of biological molecules. Lipids, nucleic acids, and proteins can serve as sensitive elements immobilized on solid phase carriers in the form of a molecular monolayer.
The SPR spectroscopy consists in the following. Two ligands** react on the surface of a special chip, one is immobilized on its surface and the other is in the solution contacting with the surface of this chip. The stream of light rays refracted at different angles is
* Plasmon resonance is stimulation of the surface plasmon (quasiparticle) at its resonance frequency by an external electromagnetic wave.--Ed.
** Ligands (Latin ligo-binding) are substances reversibly forming complexes with each other.--Ed.
Science in Russia, No. 1, 2012
Surface plasmon resonance spectroscopy-principle of the method.
Cross section of the biosensor solid phase carrier with the immobilized receptor.
focused on this surface. At a certain angle the chip's surface (usually very fine gold or silver film) absorbs this light. Binding of the ligand to it changes its absorption characteristics, which leads to modification of the initial value of the preset angle. The changes proper are permanently recorded by the SPR spectrometer and are expressed in resonance units (RU) in direct proportion to the amount of the bound ligand.
At present this analytical method is widely used for monitoring of various surface interactions, including the antibody-antigen*, protein-nucleic acids, DNA hybridization, and RNA adsorption of minor molecules, etc. Direct measurements of intermolecular interactions by SPR spectroscopy are more sensitive than measurements by many traditional methods. One more advantage of the new technology is its high speed--the studies are carried out several times more rapidly.
However, despite its efficiency, SPR spectroscopy (due to some limitations) was not widely used for studies of GPCR up to the present time. Fortunately, on the basis of effective solution of some methodological problems in recent years, the limitations have been removed and the method will be used in wide-scale screenings to find new drugs in the near future.
* Antibodies (immunoglobulins) are soluble complex glycoproteins present in the blood serum, interstitial fluid, or on the cellular membrane; they recognize and bind antigens--alien substances harmful for the host.--Ed.
Receptor binding signal on a biosensor with ligand.
SEARCH FOR A PROTOTYPE
It is noteworthy that correct arrangement and stability of membrane receptors on the carrier surface is a necessary condition for the use of biosensor solid phase carriers for studies of properties of these receptors. One more important factor is receptor fixation in the form of an inert homogeneous monolayer, as this allows stimulation directly on the biosensor surface, which opens up the possibility of studying signal transduction (transformation and amplification) processes in the real-time mode. And-the last but not the least-it should be possible to use the system repeatedly, that is, to carry out repeated immobilization and analysis cycles, for which it is essential to develop a method for regeneration of an activated receptor on the biosensor surface.
Not long ago we have developed a new method for immobilization of a visual receptor rhodopsin*, a typical representative of the GPCR family on the solid phase carrier. The component reversibly binding this protein by noncovalent interactions was applied onto the matrix. Monoclonal antibodies were used as such binding component. However, it was then found that this sensor could not be used repeatedly for immobilization and analysis. The correct structure of antibodies was impaired after 5-8 cycles and the receptor layer could not be restored. So, another method was chosen as the main one, based on rhodopsin interaction with concavalin (ConA) lectin**. ConA was covalently binded to the carrier matrix and molecules of dark inert rhodopsin were "fixed" on the resultant monolayer orientated so as to make the receptor cytoplasmatic domain available for interactions with the trans-ducin G-protein, involved in transmission of photosignal from the cell surface. The capacity of the resultant biosensor was tested by means of SPR spectroscopy. Transducin supply in the darkness did not lead to amplification of the SPR signal, which indicates the interaction absence of ligands. However, laser exposure at a wavelength of 532 nm induced a significant stimulation of the SPR signal, indicating photostimulation of rhodopsin molecules on the biosensor surface and transducin binding to them. As this biosensor not only worked correctly, but was also highly stable, was capable of repeated regeneration (>50 cycles), the above-described technology of its design was acknowledged as effective.
We intend to use this approach for immobilization and studies of mutant rhodopsin forms associated with ocular diseases*. The use of this technology opens up new vistas for the creation of biosensors of this kind based on other receptors conjugated with G-proteins. This will allow in the near future to carry out screening of numerous molecules for detection of perspective drugs with preset characteristics, with the G-protein conjugated receptors as targets.
* Rhodopsin (visual purple) is a photosensitive complex protein, the main visual pigment of the stab cells of the eye retina in vertebrates and humans.--Ed.
** Lectins are proteins capable of specific reversible binding of carbohydrates in biopolymers; concavalin is soluble lectin.--Ed.
* See: I. Senin, V. Yerichev, V, Skulachev, "Mitoengineering in Ophthalmology", Science in Russia, No. 2, 2011--Ed.
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